CN105219697A - A kind of people's primary keratinocyte is separated the method for preparation - Google Patents
A kind of people's primary keratinocyte is separated the method for preparation Download PDFInfo
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- CN105219697A CN105219697A CN201410321577.2A CN201410321577A CN105219697A CN 105219697 A CN105219697 A CN 105219697A CN 201410321577 A CN201410321577 A CN 201410321577A CN 105219697 A CN105219697 A CN 105219697A
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Abstract
The invention discloses a kind of method that people's primary keratinocyte is separated preparation, it comprises the following steps: (1) primary keratinocyte is separated; (2) primary keratinocyte is cultivated; (3) primary keratinocyte is frozen; (4) primary keratinocyte Activity determination.Keratinocyte provided by the invention is separated the method for preparation, adopt the special culture system of serum-free keratinocyte that DKSFM composition limits, not containing bovine serum composition and trophocyte, avoid the introducing of inhuman source activity albumen and other biotic component, make downstream research system more single; Direct frozen primary cell, after recovery, cell has higher survival rate, serum-free keratinocyte special culture media simultaneously owing to adopting composition to limit, decrease the factor content promoting cytodifferentiation, be conducive to the prolongation in keratinocyte vitro culture cycle, show more doublings, make downstream research system more stable.
Description
Technical field
The present invention relates to a kind of method that people's primary keratinocyte is separated preparation.
Background technology
Human keratinized cell is the main cell of group adult epidermal, because the epidermal sheet cultivated may be used for various skin disease such as clinical transplantation treatment burn, vitiligo etc., obtain people's epidermal keratinocyte of stablizing high viability for basic and clinical study is all very important.Keratinocyte separation general at present and culture system can introduce the composition in inhuman source mostly, such as use serum and trophocyte, greatly bring security risk.Keratinocyte is a kind of primary cell of the very easy differentiation of end eventually simultaneously, and as research object, often needs reasonable stability and passage capacity, and this improves separation and culture system with regard to needs.
Summary of the invention
The technical problem to be solved in the present invention is the defect overcoming prior art, provides a kind of keratinocyte to be separated the method for preparation.
In order to solve the problems of the technologies described above, the invention provides following technical scheme:
A kind of people's primary keratinocyte of the present invention is separated the method for preparation, and it comprises the following steps:
(1) primary keratinocyte is separated: get fresh skin sample through ethanol disinfection, remove skin surface microbiological contamination, clean several times with phosphate buffered saline buffer afterwards, remove the residual ethanol on sample, sample is cut into the molecule of 1.8 ~ 2.2mm diameter; With the collagen protein ferment treatment gained molecule that the work of 35 ~ 45mL enzyme is 150-300U/mL, 4 DEG C of placements of spending the night; Collecting by filtration containing the face tissue of keratinocyte, and puts into pancreas enzyme-EDTA 35 ~ 40 DEG C process 1-3 hour, separate keratinocytes; Centrifugal collecting cell precipitation under rotating speed is 1800rpm; With 40mLDMEM cell culture medium washed cell 2 times; The skin cells keratinocyte special culture solution DKSFM collected is resuspended, uses blood-counter system to calculate cell quantity and activity; According to 5 × 10
4cells/cm
2being seeded in diameter is 100mm Tissue Culture Dish;
(2) primary keratinocyte is cultivated: latter 2nd day of inoculation, observation of cell adherent growth state under inverted microscope, record growth conditions; After cell inoculation, every day changes fresh culture; Cell inoculation observed and recorded cell attachment growing state rear every day;
(3) primary keratinocyte is frozen: the degree of converging reaching 60-90% after cell inoculation culture 3-7 days, discards substratum, clean several times with phosphate buffered saline buffer; With 5mLTrypLE process cell, at 37 DEG C, 5-10 minute; Basis of microscopic observation cellular contraction situation, become after circle until all cells and add 10mLDKSFM, slight piping and druming makes cell dispersal become suspension, takes out 100ul cell and carries out cell counting, centrifugal collecting cell precipitation under 1800rpm; 90%DKSFM+10%DMSO configures cells frozen storing liquid, according to cell counts, according to 1 × 10
6cells/mL adds cells frozen storing liquid, and re-suspended cell inserts Programmed cryopreservation box, puts into subzero 80 DEG C of Ultralow Temperature Freezer frozen overnight cells; From upper step freezing storing box, take out cell, put into the medium-term and long-term preservation of liquid nitrogen container, and record preserve batch and quantity;
(4) primary keratinocyte Activity determination: choose arbitrarily 1 pipe from a collection of frozen primary keratinocyte, cell is melted rapidly in 37 C water bath, add not containing antibiotic DKSFM cell culture fluid, take out the calculating that 100ul carries out cell quantity and survival rate; Cell suspension 1800rpm is centrifugal, and collecting cell precipitates; DKSFM re-suspended cell, makes cell dispersed, inoculating cell culture dish; The CO of 5% is adopted afterwards at 37 DEG C
2cultivate; Example of spatial compartmentalizationis, when reaching 40-60% to cell confluency degree, changes replaced medium every day into; Every day is by inverted microscope observation of cell growth conditions and whether bacteriological infection situation, and Taking Pictures recording; When cell confluency degree reaches 60-90%, passage cell; Cell continues to be cultured to and breaks up completely, when stopping propagation, according to initiator cell quantity when final cell quantity and recovery, calculates multiplication cycle and number of times.
The beneficial effect that the present invention reaches is:
(1) the special culture system of serum-free keratinocyte adopting DKSFM (Definedkeratinocyte-SFM) composition to limit, not containing bovine serum composition and trophocyte, avoid the introducing of inhuman source activity albumen and other biotic component, make downstream research system more single;
(2) direct frozen primary cell, after recovery, cell has higher survival rate, can up to more than 75%, serum-free keratinocyte special culture media simultaneously owing to adopting composition to limit, decrease the factor content promoting cytodifferentiation, be conducive to the prolongation in keratinocyte vitro culture cycle, show more doublings, be greater than 26 times, make downstream research system more stable.
Embodiment
Below the preferred embodiments of the present invention are described, should be appreciated that preferred embodiment described herein is only for instruction and explanation of the present invention, is not intended to limit the present invention.
A kind of keratinocyte of the present invention is separated the method for preparation, and it comprises the following steps:
(1) primary keratinocyte is separated: get fresh postoperative dermatological specimens of peritomizing through ethanol disinfection, remove skin surface microbiological contamination, clean several times with phosphate buffered saline buffer afterwards, remove the residual ethanol on sample, sample is cut into the molecule of 2mm diameter; With the collagen protein ferment treatment gained molecule that the work of 35 ~ 45mL enzyme is 150-300U/mL, 4 DEG C of placements of spending the night; Collecting by filtration containing the face tissue of keratinocyte, and puts into pancreas enzyme-EDTA 35 DEG C process 1 hour, separate keratinocytes; Centrifugal collecting cell precipitation under rotating speed is 1800rpm; With 40mLDMEM (high sugar) cell culture medium washed cell 2 times; The skin cells collected is (containing a large amount of keratinocyte, a small amount of melanophore and inoblast etc.) use keratinocyte special culture solution DKSFM (Gibco, ThermoFisher, USA) resuspended, use blood-counter system to calculate cell quantity and activity; According to 5 × 10
4cells/cm
2being seeded in diameter is 100mm Tissue Culture Dish (Corning, USA);
(2) primary keratinocyte is cultivated: within the 2nd day, rise after inoculation, observe and record cell attachment growth conditions under inverted microscope; After cell inoculation, every day changes fresh culture; Cell inoculation observed and recorded cell attachment growing state rear every day;
(3) primary keratinocyte is frozen: reach the degree of converging of 60% after cell inoculation culture 3-7 days, discard substratum, clean several times with phosphate buffered saline buffer; Cell is processed with 5mLTrypLE (Gibco, ThermoFisher, USA), at 37 DEG C, 5-10 minute; Basis of microscopic observation cellular contraction situation, become after circle until all cells and add 10mLDKSFM, slight piping and druming makes cell dispersal become suspension, takes out 100ul cell and carries out cell counting, centrifugal collecting cell precipitation under 1800rpm; 90%DKSFM+10%DMSO configures cells frozen storing liquid, according to cell counts, according to 1X10
6cells/mL adds cells frozen storing liquid, and re-suspended cell inserts Programmed cryopreservation box, puts into subzero 80 DEG C of Ultralow Temperature Freezer frozen overnight cells; From upper step freezing storing box, take out cell, put into the medium-term and long-term preservation of liquid nitrogen container, and record preserve batch and quantity;
(4) primary keratinocyte Activity determination: choose arbitrarily 1 pipe from a collection of frozen primary keratinocyte, cell is melted rapidly in 37 C water bath, add not containing antibiotic DKSFM cell culture fluid, take out the calculating (viable cell/total cellular score) that 100ul carries out cell quantity and survival rate; Cell suspension 1800rpm is centrifugal, and collecting cell precipitates; DKSFM re-suspended cell, makes cell dispersed, inoculating cell culture dish; The CO of 5% is adopted afterwards at 37 DEG C
2cultivate; Example of spatial compartmentalizationis, when reaching 40-60% to cell confluency degree, changes replaced medium every day into; Every day is by inverted microscope observation of cell growth conditions and whether bacteriological infection situation, and Taking Pictures recording; When cell confluency degree reaches 60%, passage cell; Cell continues to be cultured to and breaks up completely, when stopping propagation, according to initiator cell quantity when final cell quantity and recovery, calculates multiplication cycle and number of times.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (1)
1. people's primary keratinocyte is separated a method for preparation, it is characterized in that, comprises the following steps:
(1) primary keratinocyte is separated: get fresh skin sample through ethanol disinfection, remove skin surface microbiological contamination, clean several times with phosphate buffered saline buffer afterwards, remove the residual ethanol on sample, sample is cut into the molecule that diameter is 2mm; With the collagen protein ferment treatment gained molecule that the work of 35 ~ 45mL enzyme is 150-300U/mL, 4 DEG C of placements of spending the night; Collecting by filtration containing the face tissue of keratinocyte, and puts into pancreas enzyme-EDTA 35 ~ 40 DEG C process 1-3 hour, separate keratinocytes; Centrifugal collecting cell precipitation under rotating speed is 1800rpm; With 40mLDMEM cell culture medium washed cell 2 times; The skin cells keratinocyte special culture solution DKSFM collected is resuspended, uses blood-counter system to calculate cell quantity and activity; Be adjusted to 5 × 10
4cells/cm
2after to be seeded in diameter be on 100mm Tissue Culture Dish;
(2) primary keratinocyte is cultivated: latter 2nd day of inoculation, observation of cell adherent growth state under inverted microscope, record growth conditions; After cell inoculation, every day changes fresh DKSFM substratum; Cell inoculation observed and recorded cell attachment growing state rear every day;
(3) primary keratinocyte is frozen: the degree of converging reaching 60-90% after cell inoculation culture 3-7 days, discards substratum, clean several times with phosphate buffered saline buffer; With 5mLTrypLE process cell, at 37 DEG C, 5-10 minute; Basis of microscopic observation cellular contraction situation, become after circle until all cells and add 10mLDKSFM, slight piping and druming makes cell dispersal become suspension, takes out 100ul cell and carries out cell counting, centrifugal collecting cell precipitation under 1800rpm; 90%DKSFM+10%DMSO configures cells frozen storing liquid, according to cell counts, according to 1 × 10
6cells/mL adds cells frozen storing liquid, and re-suspended cell inserts Programmed cryopreservation box, puts into subzero 80 DEG C of Ultralow Temperature Freezer frozen overnight cells; From upper step freezing storing box, take out cell, put into the medium-term and long-term preservation of liquid nitrogen container, and record preserve batch and quantity;
(4) primary keratinocyte Activity determination: choose arbitrarily 1 pipe from a collection of frozen primary keratinocyte, cell is melted rapidly in 37 C water bath, add not containing antibiotic DKSFM cell culture fluid, take out the calculating that 100ul carries out cell quantity and survival rate; Cell suspension 1800rpm is centrifugal, and collecting cell precipitates; DKSFM re-suspended cell, makes cell dispersed, inoculating cell culture dish; The CO of 5% is adopted afterwards at 37 DEG C
2cultivate; Example of spatial compartmentalizationis, when reaching 40-60% to cell confluency degree, changes replaced medium every day into; Every day is by inverted microscope observation of cell growth conditions and whether bacteriological infection situation, and Taking Pictures recording; When cell confluency degree reaches 60-90%, passage cell; Cell continues to be cultured to and breaks up completely, when stopping propagation, according to initiator cell quantity when final cell quantity and recovery, calculates multiplication cycle and number of times.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115354015A (en) * | 2022-07-13 | 2022-11-18 | 北京康曦健康医学研究院 | Method for adherent culture of human epidermal keratinocytes by using electromagnetic field |
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| US4940666A (en) * | 1983-07-15 | 1990-07-10 | University Patents, Inc. | Process and defined medium for growth of human epidermal keratinocyte cells |
| WO1999054435A2 (en) * | 1998-04-17 | 1999-10-28 | Societe Des Produits Nestle | Immortalised cell lines derived from normal human skin tissues |
| CN1560235A (en) * | 2004-02-17 | 2005-01-05 | 华东理工大学 | Non serum substratum for in vitro culture and amplification of cutaneous keratin cell |
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| CN1571835A (en) * | 2001-10-17 | 2005-01-26 | 贝林格尔英格海姆法玛两合公司 | Keratinocytes which may be used as biologically active substances for the treatment of wounds |
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| US4940666A (en) * | 1983-07-15 | 1990-07-10 | University Patents, Inc. | Process and defined medium for growth of human epidermal keratinocyte cells |
| WO1999054435A2 (en) * | 1998-04-17 | 1999-10-28 | Societe Des Produits Nestle | Immortalised cell lines derived from normal human skin tissues |
| CN1571835A (en) * | 2001-10-17 | 2005-01-26 | 贝林格尔英格海姆法玛两合公司 | Keratinocytes which may be used as biologically active substances for the treatment of wounds |
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| CN1562392A (en) * | 2004-03-24 | 2005-01-12 | 华东理工大学 | Method for fabricating activated artificial skin tissue in bilayer by using bioreactor |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115354015A (en) * | 2022-07-13 | 2022-11-18 | 北京康曦健康医学研究院 | Method for adherent culture of human epidermal keratinocytes by using electromagnetic field |
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Application publication date: 20160106 |